Researchers have created a new imaging compound in mice that selectively
binds to certain cancer cells and glows, or fluoresces, only when
processed by these cells. This cancer-specific fluorescence allowed
the investigators to successfully visualize very small tumors in
the peritoneum — the tissue that lines the wall of the abdomen — in
mice with ovarian cancer. The sensitivity — or ability to
accurately detect small clusters of tumor cells — of this
approach was 92 percent. The study, conducted by researchers at
the National Cancer Institute (NCI), part of the National Institutes
of Health (NIH), and colleagues, appears in the March 15, 2007
issue of Cancer Research.

“The virtue of this study is that other fluorescent compounds
have been tested for the detection of small clusters of cancer
cells that might otherwise be missed during surgery, but those
have drawbacks, including being always fluorescent thereby making
it difficult to distinguish tumor cells from normal tissue. This
study points to a potential solution to this problem,” said NIH
Director Elias A. Zerhouni, M.D.

“A fluorescent imaging compound that is specific for cancer cells
holds great promise for the treatment of cancers, such as ovarian
and pancreatic cancer, which often metastasize widely before diagnosis.
In the coming years, as cancer research is increasingly based on
an understanding of tumors down to a detailed molecular level,
advanced imaging will be a key component of essentially every study,” said
NCI Director John E. Niederhuber, M.D.

The researchers, led by Hisataka Kobayashi, M.D., Ph.D., from
NCI’s Molecular Imaging Program in the Center for Cancer Research,
created a compound to be tested only in mice that consisted of
the protein avidin, which binds to another protein commonly found
on the surface of cancer cells that potentially can spread, or
metastasize, to the peritoneum. They joined this compound to three
molecules of the fluorescent compound rhodamine X. In this new
compound, which they called Av-3ROX, the rhodamine X molecules
are unable to fluoresce. However, when Av-3ROX is taken up by cancer
cells after binding to them, it is broken down in sac-like compartments
inside the cells called lysosomes. When enzymes in the lysosomes
break the compound into smaller pieces, the rhodamine X is released
and is able to fluoresce.

“Conventional imaging methods such as nuclear isotopes, MRI, or
CT use contrast agents that make a signal whether they are bound
or unbound to a cancer cell,” said Kobayashi. “Our method will
make a signal only from cancer cells. It’s cancer-specific imaging.”

When the researchers injected the ‘always on’ fluorescent molecule
Av-0.5ROX into the peritoneum of tumor-bearing mice, fluorescence
was immediately detectable and more intense than that produced
by Av-3ROX immediately following its injection. However, Av-0.5ROX
produced fluorescence in both tumor cells and the surrounding tissue,
making it difficult to distinguish the tumor cells. In contrast,
by three hours after Av-3ROX injection, the fluorescence intensity
in normal tissues was less than with Av-0.5ROX , but the fluorescence
intensity in tumor nodules was much higher than with Av-0.5ROX.

To confirm that Av-3ROX was primarily processed by tumor cells,
the researchers performed a second experiment in mice, this time
using cells that carried the gene for red fluorescent protein (RFP)
to induce the initial tumors and peritoneal metastases. This approach
allowed every metastasis to be detected using a camera and filter
specific for RFP. The investigators then injected Av-3ROX into
the peritoneum of the mice and captured fluorescent images of both
Av-3ROX and RFP. Next, they compared the number of metastases identified
using both compounds.

Out of 507 metastases, at least 0.8 millimeters in diameter, shown
by RFP, Av-3ROX detected 465 of them, indicating a sensitivity
of 92 percent. Only 2 percent of metastases identified by Av-3ROX
turned out to be false positives, translating to a 98-percent tumor
detection accuracy, or specificity, for this technique.

Although the data provide proof-of-concept for this type of molecular
imaging technique, Av-3ROX cannot be used in people, because the
avidin portion of the compound would cause an immune system reaction.
Kobayashi and his colleagues are now working on a second-generation
compound that joins the binding site of avidin — the part
that recognizes the cancer cells — to human serum albumin.
This compound “should not create a harmful immune response because
it’s based on a human protein,” said Kobayashi.

The authors believe that this approach to molecular imaging holds
promise as a method of optically enhancing surgical or endoscopic
procedures, allowing for more complete surgical removal of metastatic
disease.

For more information about cancer, please visit the NCI website
at http://www.cancer.gov, or
call NCI's Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

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Human Services. It is the primary federal agency for conducting
and supporting basic, clinical and translational medical research,
and it investigates the causes, treatments, and cures for both
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